Novel Observation That Mice Lacking the Fifth Complement Cascade Protein Component (C5) Are Very Poor Stem Cell Mobilizers Explained by Defective Egress of Granulocytes: A Novel Role for Bone Marrow Granulocytes to Act as “ice Breaker” Cells in Facilitating Egress of Hematopoietic Stem/Progenitor Cells

Abstract

We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway and, as result of this, several potent bioactive CC anaphylatoxins (C3a, desArgC3a, C5a and desArgC5a) are released (Blood 2003; 101,3784; Blood 2004; 103,2071; Blood 2005; 105,40). Bioactive CC anaphylatoxins (C5a and desArgC5a) are also potent chemoattractants of granulocytes that bind to G-protein-coupled, seven trans-membrane span C5a receptors (C5aR and C5L2) on these cells. To learn more on the role of C5 cleavage fragments in HSPC mobilization, we studied mobilization in C5−/− and C5aR−/− mice as well as their normal wildtype littermates. Mobilization was induced by granulocyte colony-stimulating factor (G-CSF; high 250 μg/kg/6 days and low dose 50 μg/kg/6 days) or zymosan (20 mg/1kg/1 hour), which activate classical and alternative pathways of CC, respectively. We evaluated mobilization efficiency by counting the number of SKL cells, colony-forming unit granulocyte-macrophages (CFU-GMs), and white blood cells circulating in peripheral blood. In parallel, we employed transmission electron microscopy (TEM) to study the morphology and integrity of BM vessels in the BM-blood barrier. Activation of CC was measured by ELISA for C3 cleavage fragments and by histochemical staining for membrane attack-complex (MAC) depositions in BM tissue. We found by ELISA and histochemistry that CC activation correlates with the level of HSPC mobilization in wildtype mice and that mobilization of HSPCs was always preceded by the release of granulocytes from BM. Thus, granulocytes are the first wave of cells that increase in number during mobilization in peripheral blood. Mobilization studies in C5−/− revealed that these animals are very poor mobilizers. TEM studies demonstrated that hematopoietic cells together with granulocytes accumulated around small vessels in the BM of C5−/− animals, but they did not migrate or cross the BM-endothelial barrier. Since C5 cleavage fragments C5a and desArgC5a are potent chemoatrractants for granulocytes but not HSPCs, we hypothesize that a lack of both these anaphylatoxins in C5−/− animals prevents egress of granulocytes from BM, which always precedes egress of HSPCs. Furthermore, in C5aR−/−, mice mobilization was normal after administration of a high optimal dose of G-CSF. However, mobilization was significantly lower after a suboptimal dose of G-CSF or administration of zymosan. This indicates that another alternative receptor for C5a and desArgC5a (C5L2) may compensate for C5aR deficiency and that it plays a role in the egress of granulocytes from the BM as well. Thus, this study demonstrates that cells from the granulocytic lineage are actively involved in mobilization in a C5a,-desArgC5a-C5aR manner not only by secreting proteases that create a proteoytic environment in BM, but also as a kind of “ice-breaker” type cells necessary for disintegration of the endothelial-BM barrier to enable HSPCs to egress from the BM microenvironment. In cases of granulocytopenia or if granulocytes are not mobilized as seen in C5−/− mutants, mobilization of HSPCs is very poor. Thus, modulation of CC activation in the BM and stimulation of granulocyte egress from the BM into circulation may help to develop more efficient strategies for HSPC mobilization.